Method For Preparing Extract From Wild Ginseng Showing Anticancer Activity And The Composition Comprising The Same

ABSTRACT

The present invention relates to a method for preparing purified extract from wild ginseng showing anticancer activity such as inhibitory of cancer cell adherence, inhibitory of cancer cell metastasis and immunostimulating effect and a composition comprising the same prepared by inventive method. The composition have potent anticancer activity, therefore, it can be used as the therapeutics for treating and preventing various cancer diseases.

TECHNICAL FIELD

The present invention relates to a method for preparing purified extractfrom wild ginseng showing anticancer activity and the compositioncomprising the same.

BACKGROUND ART

Cancer is a malignant tumor occurred by the disorder in cell cyclingresulting in abnormal differentiation and develops through three steps,i.e., initiation, promotion, and progression. The initiation of cancercan be occurred by sufficient amount of carcinogenic substance mostly,however, small amount of initiator gives rise to cell mutating normalcell, proliferating mutated cells and finally stimulating tumor promotercausing to promote the differentiation of abnormal cells resulting inthe formation of cancer tissue.

There have been numerous attempts or methods to develop an anticancerdrug to treat various cancer till now, for example, a method forscreening a cytotoxic substance acting on cancer cell directly, a methodfor screening an modulating substance of the immunity of body, a methodfor screening an inhibiting substance of cancer cell metastasis, amethod for screening an inhibiting substance of angiogenesis having beenintensively studied recently and so on.

Conventionally used anticancer drug may be classified into three groups:i.e., biological drugs such as gene or enzyme preparation, vaccine etc;chemotherapic agent (pure synthetic anticancer drug) such as taxol,vinblastine, etc; and natural product drug derived from naturalresources. However, the biological drugs have not yet reached to beclinically used. Moreover, the chemotherapic agents have limit to usebecause of their various adverse actions such as the occurrence ofintolerance to specific anticancer agent, a malfunction of bone marrow,stomach disorder such as vomiting, nausea, hair loss in spite of theirpotent anticancer activities (Gillman et al., Maxwell Macmillan., 18, pp1202, 1986; Chung et al., J. Wonkwang Medical Sci., 3, pp 13-34, 1987).There have been reported that since most of anticancer drugs have lowmolecular weight of anticancer drug, the anticancer drug can permeatenormal cell, especially actively differentiating cell, as well as cancercell resulting in the damage of normal cell and it is easily excretedfrom urea, which requires relative amount of drugs (Yamazaki et al.,Biosci. Biotech. Biochem., 56(1), pp 149, 1992; Tompson et al., Exp.Cell Res., 41, pp 411-427, 1966; Ellem et al, Devel. Biol., 118, pp311-330, 1968).

Recently, there have been lots of attempts to develop anticancer agentshaving preventing or treating cancer diseases from crude drug or naturalproduct.

Accordingly, there have been urgently needed to find effectivesubstances providing with verified efficacy as well as low or at leasttoxicity from natural resource till now. Recently, alternative medicine,especially, Chinese medical therapy based on immune potentiatingmechanism has been highlighted as an alternative method withconventional Western medicine to deviate the adverse effect ofchemotherapy. Among the Chinese medical therapy, together with a use ofplant extract extracted from Chinese drug showing immune potentiatingactivity and less toxicity, a use of acupuncture treatment witheffective extract become highlighted in Korea, which comprises the stepsinsisting of: selecting plant or other natural resource having mostpotent effect on individual disease; extracting effective ingredientfrom the extract providing with maximized efficacy and minimizedtoxicity; inoculating or injecting the ingredient into the spotssuitable for acupuncture or painful spots on the body and therefore itcould endow with synergic effect due to the effect of acupuncture andthe pharmacological effect of the ingredient. However, there have beenneeded to obtain more purified extract having less toxicity than crudeform which could express its toxicity in administrating into injectionor acupuncture such as fever, pain, edema etc.

There has been not reported or disclosed about a method for preparingpurified extract from wild ginseng showing anticancer activity and thecomposition comprising the same in any of above cited literatures, thedisclosures of which are incorporated herein by reference.

To investigate an effect of purified extract from wild ginseng preparedby the method of the present invention on the cancer and tumor cells,the inventors of the present invention have intensively carried outseveral in vitro and in vivo model experiments, and finally completedpresent invention by confirming that the purified extract showsanticancer activity and immunostimulating effect.

These and other objects of the present invention will become apparentfrom the detailed disclosure of the present invention providedhereinafter.

DISCLOSURE

The present invention provides a method for preparing purified extractfrom wild ginseng having treating and preventing activity of cancerdisease.

The present invention provides a pharmaceutical composition comprising apurified extract of wild ginseng prepared from above described method asan active ingredient in an effective amount to treat and prevent cancerdisease by anticancer activity.

The present invention also provides a use of above extract for thepreparation of pharmaceutical composition to treat and prevent cancerdisease by anticancer activity in mammal or human.

Accordingly, it is an object of the present invention to provide amethod for preparing a purified extract from wild ginseng comprising thesteps insisting of; subjecting the wild ginseng material to distillationextracting method with extracting solvent repeatedly; freezing thedistilled extract to obtain frozen purified extract; thawing the extractand filtrating to obtain purified extract.

It is an object of the present invention to provide a pharmaceuticalcomposition comprising a purified extract of wild ginseng prepared byabove described method as an active ingredient in an effective amount totreat and prevent cancer disease.

It is an object of the present invention to provide a use of a purifiedextract of wild ginseng prepared by above described method for thepreparation of therapeutic agent for the treatment and prevention ofcancer disease in mammal or human.

It is an object of the present invention to provide a method of treatingor preventing cancer disease in mammal or human comprising administeringto said mammal or human an effective amount of a purified extract ofwild ginseng prepared by above described method, together with apharmaceutically acceptable carrier thereof.

The term disclosed herein ‘extracting solvent’ comprises water, loweralcohols such as methanol, ethanol, preferably water.

The term disclosed herein ‘wild ginseng material’ comprises all the wildginseng cultivated or naturally grown wild ginseng in the world, forexample, Korea, Japan, Russia, China, Europe, America etc.

The pharmaceutical composition of the present invention can containabout 0.01-50% by weight of the above extract based on the total weightof the composition.

An inventive a purified extract of wild ginseng may be prepared inaccordance with the following preferred embodiment.

Hereinafter, the present invention is described in detail.

An inventive purified extract of wild ginseng can be prepared in detailby following procedures,

The inventive purified extract of wild ginseng can be prepared byfollows;

Specifically, it is an object of the present invention to provide amethod for preparing a purified extract from wild ginseng comprising thesteps insisting of; subjecting the wild ginseng material to the 1^(st)distillation extracting method with extracting solvent to obtain the1^(st) distillate at the 1^(st) step; subjecting the 1^(st) distillateto the 2^(nd) distillation extracting method with extracting solvent toobtain the 2^(nd) distillate at the 2^(nd) step; freezing the 2^(nd)distillate to obtain frozen purified extract at the 3^(rd) step; thawingthe extract and filtrating to obtain filtrated extract at the 4^(th)step; heating the filtrate with double boiler and refreezing at the5^(th) step; thawing and sterilizing the extract to obtain the purifiedextract of the present invention at the 6^(th) step.

Specifically, At the 1^(st) step, it is preferable that the wild ginsengmaterial is poured to polar solvent selected from water, lower alcoholsuch as methanol, ethanol, propanol and the solvent mixture there ofpreferably, water, more preferably, water in the ratio ranging from 1.0kg to 3.0 kg of material per liter of water and left alone at roomtemperature for the period ranging from 1 to 4 hours, preferably, 3hours. Subsequently, it is heated with distillation apparatus at thetemperature ranging from 60 to 120° C., preferably 80 to 100° C., morepreferably, 100° C., for the period ranging from 6 to 24 hours,preferably, from 8 to 10 hours gradually to obtain the 1^(st)distillate.

At the 2^(nd) step, it is preferable that the 1^(st) distillate is addedto the boiling chip containing flask equipped with distillationapparatus and heated at the temperature ranging from 60 to 120° C.,preferably 80 to 100° C., more preferably, 100° C., for the periodranging from 7 to 24 hours, preferably, from 8 to 12 hours gradually andthe heating process is maintained to the extent that the volume ofconcentrate is reduced to be in the ranging 70 to 90 (v/v) % to obtainthe 2^(nd) distillate.

At the 3^(rd) step, it is preferable that the 2^(nd) distillate issubjected to freezing process at the temperature ranging from −5 to −15°C., preferably from −8 to −12° C. and the process is maintained to theextent that the volume of un-frozen fraction is decreased to be lessthan 5% of the total volume. The un-frozen fraction is removed and theremaining distillate is subjecting to thawing process. Those freezingand removing un-frozen fraction processes can be repeated, preferably 1to 6 times, to obtain frozen purified extract of the present invention.Through the 3^(rd) step, toxic substance in wild ginseng material at the1^(st) step is almost or completely removed in purified extract preparedby this step.

At the 4^(th) step, it is preferable that the frozen purified extract atthe 3^(rd) step is subjected to thawing process at room temperature andfiltrating process is followed with filter paper having pore sizeranging from 0.1 to 0.6 um to obtain filtrates.

At the 5^(th) step, the filtrate prepared in above step is heated withdouble boiler at the temperature ranging from 60 to 120° C., preferably80 to 100° C., in the period ranging from 20 to 40 mins, preferably, 30mins, and is refrozen completely at the temperature ranging from −30 to−10° C., preferably, at −15° C. to obtain frozen purified extract.

At the 6^(th) step, the frozen filtrate is thawed and sterilized toobtain final purified extract of the present invention.

In above described the 1^(st) step, the concentration of the 1^(st)distillate can be controlled by changing the ratio of material pursuantto the purpose of the present invention.

In above described purification process, the extract can be separatedinto two parts, i.e., frozen part and unfrozen part according to thedifference with specific ingredients. Since the ingredients havingstronger toxicity being contained in the extract become frozen slowerthan those having weaker toxicity because of their low freezing point,the unfrozen part containing most of toxic ingredient can be removed byrepeating the above freezing processes,

Accordingly, it is an object of the present invention to provide apharmaceutical composition comprising a purified extract of wild ginsengprepared by above described method as an active ingredient in aneffective amount to treat and prevent cancer disease by anticanceractivity.

It is an object of the present invention to provide a use of a purifiedextract of wild ginseng prepared by above described method for thepreparation of therapeutic agent for the treatment and prevention ofcancer disease in human or mammal.

It is an object of the present invention to provide a method of treatingor preventing cancer disease in a mammal or human comprisingadministering to said mammal or human an effective amount of a purifiedextract of wild ginseng prepared by above described method, togetherwith a pharmaceutically acceptable carrier thereof.

Through in vitro and in vivo model experiments to infirm the effect ofthe purified extract of the present invention on the cancer and tumorcells, present inventors infirm that the purified extract showsanticancer activity and immunostimulating effect.

Therefore, the purified extract of the present invention can be usefulin treating and preventing cancer disease.

In addition to the efficacy, the purified extract of the presentinvention can be used safely in long-term administration since it hasbeen used as a commercial crude drug since long years ago.

The inventive imposition for treating and preventing cancer disease maycomprises above extracts as 0.01˜50% by weight based on the total weightof the composition.

The inventive composition may additionally comprise conventionalcarrier, adjuvants or diluents in accordance with a using method wellknown in the art. It is preferable that said carrier is used asappropriate substance according to the usage and application method, butit is not limited. Appropriate diluents are listed in the written textof Remington's Pharmaceutical Science (Mack Publishing co, Easton Pa.).

Hereinafter, the following formulation methods and excipients are merelyexemplary and in no way limit the invention.

The composition according to the present invention can be provided as apharmaceutical composition containing pharmaceutically acceptablecarriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose,sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acaciarubber, alginate, gelatin, calcium phosphate, calcium silicate,cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxy benzoate, talc, magnesium stearate and mineraloil. The formulations may additionally include fillers,anti-agglutinating agents, lubricating agents, wetting agents, flavoringagents, emulsifiers, preservatives and the like. The compositions of theinvention may be formulated so as to provide quick, sustained or delayedrelease of the active ingredient after their administration to a patientby employing any of the procedures well known in the art.

For example, the compositions of the present invention can be dissolvedin oils, propylene glycol or other solvents that are (mainly used toproduce an injection. Suitable examples of the carriers includephysiological saline, polyethylene glycol, ethanol, vegetable oils,isopropyl myristate, etc., but are not limited to them. For topicaladministration, the extract of the present invention can be formulatedin the form of ointments and creams.

Pharmaceutical formulations containing present composition may beprepared in any form, such as oral dosage form (powder, tablet, capsule,soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet,granule), or topical preparation (cream, ointment, lotion, gel, balm,patch, paste, spray solution, aerosol and the like), injectablepreparation (solution, suspension, emulsion) or acupuncture injectablepreparation.

The composition of the present invention in pharmaceutical dosage formsmay be used in the form of their pharmaceutically acceptable salts, andalso may be used alone or in appropriate association, as well as incombination with other pharmaceutically active compounds.

The desirable dose of the inventive extract or composition variesdepending on the condition and the weight of the subject, severity, drugform, route and period of administration, and may be chosen by thoseskilled in the art. However, in order to obtain desirable effects, it isgenerally recommended to administer at the amount ranging 10 g/kg,preferably, 1 to 3 g/kg by weight/day of the inventive extract orcompounds of the present invention. The dose may be administered insingle or divided into several times per day. In terms of composition,the amount of inventive extract should be present between 0.01 to 50% byweight, preferably 0.5 to 40% by weight based on the total weight of thecomposition.

The pharmaceutical composition of present invention can be administeredto a subject animal such as mammals (rat, mouse, domestic animals orhuman) via various routes. All modes of administration are contemplated,for example, administration can be made orally, rectally or byintravenous, intramuscular, subcutaneous, intracutaneous, intrathecal,epidural or intracerebroventricular injection or acupuncture injectiononto the spots suitable for acupuncture.

Inventive extract of the present invention have no toxicity and adverseeffect therefore; they can be used with safe.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the compositions, use andpreparations of the present invention without departing from the spiritor scope of the invention.

DESCRIPTION OF DRAWINGS

The above and other objects, features and other advantages of thepresent invention will more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which;

FIG. 1 shows HPLC analyzing un purified extract of naturally grown wildginseng;

FIG. 2 shows HPLC analyzing the purified extract of naturally grown wildginseng.

MODE FOR INVENTION

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the compositions, use andpreparations of the present invention without departing from the spiritor scope of the invention.

The present invention is more specifically explained by the followingexamples.

However, it should be understood that the present invention is notlimited to these examples in any manner.

The following Reference Example, Examples and Experimental Examples areintended to further illustrate the present invention without limitingits scope.

Comparative Example 1 Preparation of Not Purified Extract of NaturallyGrown Wild Ginseng

2.5 kg of Sanyang wild ginseng originated from naturally grown wildginseng was washed with salt water and sliced into piece at the width of3 nm, poured into flask containing 1 liter of water and heated at 100°C. for eight. The distillate was filtered with filter paper having apore size ranging from 0.1 to 0.6 um. The filtrate was soled at roamtemperature, sterilized and stored in sterilized bottle maintaining thetemperature at below 4° C. to use as a sample in following experiments.

Comparative Example 2 Preparation of Not Purified Extract of CultivatedWild Ginseng

2 kg of cultivated wild ginseng originated from naturally grown wildginseng was washed with salt water and sliced into piece at the width of3 nm, poured into flask containing 1 liter of water and heated at 100°C. for eight. The distillate was filtered with filter paper having apore size ranging from 0.1 to 0.6 um. The filtrate was soled at roamtemperature, sterilized and stored in sterilized bottle maintaining thetemperature at below 4° C. to use as a sample in following experiments.

Example 1 Preparation of the Purified Extract of Naturally Grown WildGinseng

2.5 kg of Sanyang wild ginseng originated from naturally grown wildginseng was washed with salt water and sliced into piece at the width of3 nm, poured into flask containing 1 liter of water and left alone forthree hours at 20° C.

The flask was equipped with distillation apparatus and heated at 100° C.for eight hours to obtain the 1^(st) distillate. 5 g of boiling chip andthe 1^(st) distillate were poured flask equipped with distillationapparatus and the flask was further heated at 100° C. for ten hours toobtain the 2^(nd) distillate. The 2^(nd) distillate was frozen to theextent that the volume of unfrozen part become less than 5% of totalvolume at −10° C. and the unfrozen part was discarded. Remaining frozenpart was thawed at room temperature. Above freezing and thawing stepswere further repeated two times. After the frozen part was thawed, itwas filtered with filter paper having a pore size of 0.1˜0.6 um and thefiltrate was heated in double boiler at 90° C. for 30 mins. The filtratewas soled and frozen completely at −15° C. The frozen purified extractwas thawed at room temperature, sterilized and stored in sterilizedbottle maintaining the temperature at below 4° C. to use as a sample infollowing experiments.

Example 2 Preparation of the Purified Extract of Cultivated Wild Ginseng

2 kg of cultivated wild ginseng supplied with Kyunghee University(Seoul, Korea) was washed with salt water and sliced into piece at thewidth of 3 nm, poured into flask containing 1 liter of water and leftalone for three hours at 20° C.

Further steps were performed with the methods similar to those inExample 1 to obtain purified extract of cultivated wild ginseng and itwas used as a sample in following experiments.

Example 3 Content Analysis of Above Comparative Example 1 and Example 1

The intent of saponin in above Comparative Example 1 and Example 1 wasdetermined by using High Performance Liquid chromatography (column:Phenomenex Nuna C18(5 um, 150×2.0 mm), flow rate: 0.3 ml/min, detector:UV detector 203 nm, developing solvent: starting solvent;CH₃CN:H₂O=15:85 and from 0% to 30% in CH₃CH:H₂O=80:20 more than 70minutes, room temperature).

As shown in FIG. 1 and FIG. 2, the result of above intent analysis wasdetected that the intent of saponin in above Comparative Example 1 andExample 1 showed a remarkable difference between examples two.

Experimental Example 1 Inhibition of Cancer Cell for Adhering toCytoplasm

If cancer cells moves from the first occurring area to other organ, thecancer cell need cell adhering ability to specific substance in thecytoplasm i.e., collagen, laminin, gelatin, etc., or blood endotheliumfor metastasis.

To determine the inhibitory effect of cell adhering ability of theextract of the present invention, MDA-MB-231 cells (1×10⁶ cells/well)were seeded in 96 well plate sated by 0.1% gelatin and variousconcentrations of Comparative Example 1, 2 and Example 1, 2 (0, 62.5,125, 250 and 500 ug/ml) were treated thereto to the concentration of 100ul/well respectively. The cells were incubated until the cells wasattached to bottom of plate at 37° C. incubator and the plate was washedwith PBS buffer cautiously. The attached cells were stained with crystalviolet staining agent and the values of OD were measured by microplatereader (ELISA reader, DENLEY Co., Japan) at 620 nm.

At the result, it is confirmed that Comparative Example 1, 2 and Example1, 2 treatment groups showed that they inhibited the adhesion ofMDA-MB-231 cells to gelatin in dose dependent manner (See Table 1).TABLE 1 Sample Concentration (μl/ml) Inhibition of adhesion (%)Comparative 62.5 13.32 ± 2.31 Example 1 125 17.21 ± 4.38 250 20.94 ±2.06 500 34.84 ± 1.04 Example 1 62.5 18.18 ± 0.74 125 22.81 ± 2.17 25029.32 ± 3.33 500 44.37 ± 5.43 Comparative 62.5 15.04 ± 3.22 Example 2125 18.90 ± 3.31 250 20.46 ± 4.46 500 25.91 ± 6.30 Example 2 62.5 17.54± 1.92 125 24.56 ± 3.31 250 32.32 ± 2.91 500 42.26 ± 3.30

Experimental Example 2 Inhibitory Effect on the Metastasis of MDA-MB-231cell

To determine the inhibitory effect of the extract of the presentinvention on cancer cell metastasis, Polycarbonate membrane having apore size of 8 um was sufficiently coated with 50 ug of matrigel for 1hour and dried in the air for 24 hours. Each 30 ul of conditioned mediasupplemented with 0.1% BSA was poured onto lower compartment of Boydenchamber. 50 ul of MDA-MB-231 cells (1×10⁶ cells/well) supplemented withRPMI1640 (FBS-free) media containing 0.1% BSA treated with 50 ul ofextract prepared by Comparative Example 1, 2 and Example 1, 2 werepoured onto upper compartment of Boyden chamber, respectively. Afterincubating for 16 hours at 37° C. in 50% CO₂ and 95% air condition in ahumidified incubator, the membrane was fixed by MeOH and stained withQuic solution (Diffco Co. Ltd). The numbers of cells invaded from uppercompartment to lower compartment were counted by optical spectroscopy.

At the result, it was confirmed that the groups treated with the extractof Example 1 and 2 showed stronger inhibition of the metastasis ofMDA-MB-231 cells in dose dependent manner compared with the groupstreated with the extract of Comparative Example 1 and 2 (See Table 2).TABLE 2 Sample Concentration (μl/ml) Inhibition rate (%) Comparative62.5 36.43 ± 3.08 Example 1 125 49.49 ± 5.85 250 50.06 ± 1.69 500 69.54± 5.35 Example 1 62.5 43.54 ± 4.85 125 59.94 ± 3.12 250 58.02 ± 3.59 50077.75 ± 6.54 Comparative 62.5 14.11 ± 2.85 Example 2 125 38.48 ± 4.22250 46.67 ± 5.27 500 48.05 ± 2.15 Example 2 62.5 38.32 ± 3.86 125 47.22± 3.05 250 58.36 ± 3.94 500 67.69 ± 4.21

Experimental Example 3 Inhibitory Effect on the Volumetric Increase ofCancer Cell Using by Lewis Lung Carcinoma Cell

To determine the inhibitory effect on the volumetric increase of cancercell, following experiment was performed by modifying the proceduredisclosed in the literature (Teruhiro et al., Cancer Res., 56, pp2809-14, 1996).

Lewis lung carcinome cells (1×10⁶ cells/well) sub-cultured in vivo ofexperimental mouse and adjusted the concentration to 1×10⁶ cells/well,were injected into the lest armpit of BDE-1 male mice (6-week-old).After 24 hours, adriamycin in the concentration of 0.5 and 1 mg/mouse,and the extracts prepared in Comparative Example 1, 2 and Example 1, 2in the concentration of 50 and 100 ul/mouse were injected into the miceintraperitoneally. The injection of samples was maintained for 2 weeksuntil the tumor volume of mice in non-treatment group used as controlgroup become to 2 cm³. After 2 weeks, the tumor volume was calculated byusing following mathematical formulae 1 and 2 to measure the inhibitionrate of tumor volume. $\begin{matrix}{{{Tumor}\quad{volume}\quad\left( {cm}^{3} \right)} = \frac{L \times w^{2}}{2}} & \left\lbrack {{Mathematical}\quad{formulae}\quad 1} \right\rbrack\end{matrix}$

L(cm): Length of the tumor

W²(cm²): Width of the tumor $\begin{matrix}{{{Inhibition}\quad{of}\quad{tumor}\quad{volume}\quad(\%)} = {\frac{A - B}{A} \times 100}} & \left\lbrack {{Mathematical}\quad{formulae}\quad 2} \right\rbrack\end{matrix}$

A Tumor volume (cm³) of control group

B Tumor volume (cm³) of sample treatment group

At the result, it was confirmed that the groups treated with the extractof Example 1 and 2 showed stronger inhibition of the volumetric growthof Lewis lung carcinoma cells in dose dependent manner compared with thegroups treated with the extract of Comparative Example 1 and 2 (SeeTable 3). TABLE 3 Sample Concentration (μl/mouse) Inhibition effect (%)Comparative 50 30.05 ± 9.47  Example 1 100 48.95 ± 2.87  Example 1 5075.94 ± 10.70 100 90 12 ± 11.25 Comparative 50 29.04 ± 10.94 Example 2100 40.38 ± 13.32 Example 2 50 73.05 ± 11.37 100 88.94 ± 10.83adriamycin 0.5 mg 31.20 ± 8.05 

Experimental Example 4 Body Weight Measurement

The body weight of mice prepared in above Experimental Example 3 wasmeasured with automatic weight measurement equipment (Jenix,Dongsintonsang, Korea) during the experimental period.

At the result, Example 1 and 2 treatment groups showed significantincrease of the body weight while most of conventionally availableanticancer drugs showed the decrease of the body weight because of theiradverse action.

Experimental Example 5 Inhibitory Effect on the Metastasis of B16-F10Melanoma Cell

To determine the inhibitory effect on the metastasis of cancer cell,B16-F10 melanoma cells (2.5×10⁵ cells/well) was injected into lateraltail vein of C57BL/6 mice. After 24 hours, the extracts prepared inComparative Example 1, 2 and Example 1, 2 in the concentration of 100ul/mouse/day were injected, respectively. After 14 days of theinjection, above experimental mice was killed and the colony of lungmetastatic tumor cell delivered from the mouse was observed bymacrography.

At the result, it was confirmed that the extract prepared in Example 1and 2 showed stronger inhibitory effect on the metastasis of B16-F10melanoma cells compared with the extracts prepared in ComparativeExample 1 and 2 (See Table 4). TABLE 4 Sample Colony number Inhibitionrate (%) Comparative Example 1 47.02 ± 13.83 42 Example 1 33.96 ± 10.4858 Comparative Example 2 65.51 ± 11.32 18 Example 2 52.23 ± 18.46 35Non-treatment  80.4 ± 17.81 0

Experimental Example 6 Immunostimulating Effect Using by SplenicLeucocytes of BALB-c Mouse

To determine the immunostimulating effect of the extract of the presentinvention, splenic leucocytes (1×10⁶ cells/ml) of BALB-c mouse weresuspended in RPMI 1640 medium (Gibco BRL Co., Ltd., USA) supplementedwith 100 ug/ml of streptomycin, 100 U/ml of penicillin and 10% fetalbovine serum, and treated with 200 ul/ml of the extracts of ComparativeExample 1, 2 and Example 1, 2, respectively. The solution was incubatedat 37° C. in 5% CO₂₀ and 95% air condition in a humidified incubator for3 days and the formation of leucocytes was determined by Flow cytometry.200 ug/ml of adriamycin treatment group was used as control group.

At the result, it was confirmed that while the adriamycin treatmentgroup decreased the immunity by 26.1%, the Example 1 and 2 treatmentgroups showed higher increased synthesis rate of lymphocytes comparedwith Comparative Example 1 and 2 treatment groups (See Table 5). TABLE 5Concentration Increase rate Sample (μg/ml) of lymphocyte (%) adriamycin200 −21.6 Comparative Example 1 200 30.6 Example 1 200 57.2 ComparativeExample 2 200 32.1 Example 2 200 54.3

Experimental Example 7 Toxicity Test

Methods (1)

The acute toxicity tests on ICR mice (mean body weight 25±5 g) andSprague-Dawley rats (235±10 g, Jung-Ang Lab Animal Inc.) were performedusing the extract of the Example 1. Four group consisting of 10 mice orrats was administrated orally intraperitoneally with 250 mg/kg, 500mg/kg, 1000 mg/kg and 5000 mg/kg of test sample or solvents (0.2 ml,i.p.) respectively and observed for 2 weeks.

Methods (2)

The acute toxicity tests on ICR mice and Sprague-Dawley rats wereperformed using the extract of the Example 2. Four group insisting of 10mice or rats was administrated intraperitoneally with 25 mg/kg, 250mg/kg, 500 mg/kg and 725 mg/kg of test sample or solvents (0.2 ml,i.p.), respectively and observed for 24 hours.

Results

There were no treatment-related effects on mortality, clinical signs,body weight changes and gross findings in any group or either gender.These results suggested that the extract prepared in the presentinvention were potent and safe.

Hereinafter, the formulating methods and kinds of excipients will bedescribed, but the present invention is not limited to them. Therepresentative preparation examples were described as follows.Preparation of powder Dried powder of Example 1 300 mg Lactose 100 mgTalc 10 mg

Powder preparation was prepared by mixing above components and fillingsealed package. Preparation of tablet Dried powder of Example 1 50 mgCorn Starch 100 mg Lactose 100 mg Magnesium Stearate 2 mg

Tablet preparation was prepared by mixing above components andentabletting. Preparation of capsule Dried powder of Example 1 50 mgCorn starch 100 mg Lactose 100 mg Magnesium Stearate 2 mg

Tablet preparation was prepared by mixing above components and fillinggelatin capsule by conventional gelatin preparation method. Preparationof injection Dried powder of Example 2 50 mg Distilled water forinjection optimum amount PH controller optimum amount

Injection preparation was prepared by dissolving active component,controlling pH to about 7.5 and then filling all the components in 2 mlample and sterilizing by conventional injection preparation method.Preparation of liquid Dried powder of Example 2 0.1˜80 g Sugar  5˜10 gCitric acid 0.05˜0.3% Caramel 0.005˜0.02%  Vitamin C   0.1˜1% Distilledwater  79˜94% CO₂ gas 0.5˜0.82%

Liquid preparation was prepared by dissolving active component, fillingall the components and sterilizing by conventional liquid preparationmethod.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the present invention, and allsuch modifications as would be obvious to one skilled in the art areintended to be included within the scope of the following claims.

INDUSTRIAL APPLICABILITY

As described in the present invention, the purified extracts of wildginseng prepared by inventive method have potent anticancer activitysuch as inhibitory of cancer cell adherence, inhibitory of cancer cellmetastasis and immunostimulating effect, therefore, it can be used asthe therapeutics for treating and preventing various cancer diseases.

1. A method for preparing a purified extract from wild ginsengcomprising the steps insisting of; subjecting the wild ginseng materialto distillation extracting method with extracting solvent repeatedly;freezing the distilled extract to obtain frozen purified extract;thawing the extract and filtrating to obtain purified extract.
 2. Themethod according to claim 1, said extracting solvent is selected fromwater, C₁-C₄ lower alcohol or the mixture thereof.
 3. The methodaccording to claim 2, said extracting solvent is water.
 4. The methodaccording to claim 1, said wild ginseng comprises cultivated ginseng andnaturally grown wild ginseng.
 5. A method for preparing a purifiedextract from wild ginseng comprising the steps insisting of; subjectingthe wild ginseng material to the 1^(st) distillation extracting methodwith extracting solvent to obtain the 1^(st) distillate at the 1^(st)step; subjecting the 1^(st) distillate to the 2^(nd) distillationextracting method with extracting solvent to obtain the 2^(nd)distillate at the 2^(nd) step; freezing the 2^(nd) distillate to obtainfrozen purified extract at the 3^(rd) step; thawing the extract andfiltrating to obtain filtrated extract at the 4^(th) step; heating thefiltrate with double boiler and refreezing at the 5^(th) step; thawingand sterilizing the extract to obtain the purified extract of thepresent invention at the 6^(th) step.
 6. The method according to claim5, said the 1^(st) step is insisting of: pouring water to said materialin the ratio ranging from 1.0 kg to 3.0 kg of material per liter ofwater; being left alone at room temperature for the period ranging from1 to 4 hours; subsequently, heating with distillation apparatus at below100° C. for the period ranging from 6 to 24 hours to obtain the 1^(st)distillate.
 7. The method according to claim 5, said the 2^(nd) step isinsisting of: adding boiling chip containing flask equipped withdistillation apparatus to the 1^(st) distillate; heating at 100° C., forthe period ranging from 7 to 24 hours gradually to the extent that thevolume of concentrate is reduced to be in the ranging 70 to 90(v/v) % toobtain the 2^(nd) distillate.
 8. The method according to claim 5, saidthe 3^(rd) step is insisting of: freezing the 2^(nd) distillate at thetemperature ranging from −5 to −15° C. to the extent that the volume ofun-frozen fraction is decreased to be less than 5% of the total volume;removing the un-frozen fraction; thawing remaining distillate; repeatingthose freezing and removing un-frozen fraction processes at 1 to 6 timesto obtain frozen purified extract of the present invention.
 9. Themethod according to claim 5, said the 4^(th) step is consisting ofthawing the frozen purified extract at the 3^(rd) step at roomtemperature; and filtrating with filter paper having pore size rangingfrom 0.1 to 0.6 um to obtain filtrates.
 10. The method according toclaim 5, said the 5^(th) step is insisting of: heating with doubleboiler at 100° C. in the period ranging from 20 to 40 mins; andrefreezing to obtain frozen purified extract.
 11. A pharmaceuticalcomposition comprising a purified extract of wild ginseng prepared bythe method as set forth in claim 1 as an active ingredient in aneffective amount to treat and prevent cancer disease by anticanceractivity.
 12. The pharmaceutical composition according to claim 11wherein said composition comprises oral dosage form such as powder,tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill,powder, sachet, granule or aerosol.
 13. The pharmaceutical compositionaccording to claim 11 wherein said composition comprises topicalpreparation such as cream, ointment, lotion, gel, balm, patch, paste,spray solution, aerosol, and injectable preparation such as acupunctureinjectable preparation.
 14. The pharmaceutical composition according toclaim 13 wherein said composition is administered via spots orally,rectally or by intravenous, intramuscular, subcutaneous, intracutaneous,intrathecal, epidural or intracerebroventricular injection oracupuncture injection onto the spots suitable for acupuncture.
 15. Thepharmaceutical composition according to claim 14 wherein saidcomposition is administered via acupuncture injection onto the spotssuitable for acupuncture.
 16. A use of a purified extract of wildginseng prepared by the method as set forth in claim 1 for thepreparation of therapeutic agent for the treatment and prevention ofcancer disease by anticancer activity in human or mammal.
 17. A methodof treating or preventing cancer disease by anticancer activity in amammal or human comprising administering to said mammal or human aneffective amount of a purified extract of wild ginseng prepared by themethod as set forth in claim 1, together with a pharmaceuticallyacceptable carrier thereof.